JP7657590B2 - Agents that increase expression of BCL2-associated cell death agonists for treating cancer - Patents.com - Google Patents

Description

The present invention relates to agents that increase the expression of tumor biomarkers for use in the treatment of cancer in combination with chemotherapeutic agents.

The success of many cancer treatments depends on their co-administration alongside adjuvant-type molecules. Without any independent therapeutic benefit, the adjuvant is responsible for stimulating the subject's immune system such that the active compound targeting the cancer can achieve maximum therapeutic effect.

Because adjuvants typically modulate a patient's immune response, they are often used in combination with cancer vaccines or biologics, such as humanized therapeutic antibodies. They act to boost the patient's immune system to increase the production of antibodies in response to cancer vaccine exposure or by suppressing or reducing the patient's immunogenicity to the foreign therapeutic antibody. Adjuvants therefore play an important role in driving immune cancer therapy toward successful therapeutic outcomes.

Frequently, immunotherapy is combined with traditional cancer treatments such as radiation therapy or chemotherapy. For certain types of cancer for which no effective immunotherapy exists, traditional treatments alone may be administered. Traditional cancer treatments may also be administered in combination, where the therapeutic effect of simultaneous administration may be greater than the sum of the effects of the individual treatments.

Despite their great efficacy, traditional cancer treatment combinations can exacerbate the side effects experienced by patients and often result in premature termination of treatment regimens. Thus, the beneficial synergistic effects of coadministering multiple anticancer drugs may not be realized due to the demanding nature of the treatments.

The development of new therapies with greater efficacy and reduced side effects could avoid the need to co-administer certain cancer therapies, thus avoiding the severe side effects that often lead to premature treatment termination. Alternatively, a similar result could be achieved by developing adjuvant-like molecules that boost the therapeutic efficacy of chemotherapy drugs.

Therefore, there is a need to develop agents that boost the therapeutic utility of chemotherapy drugs to minimize the deleterious side effects of what would otherwise be aggressive cancer treatment regimens.

The present inventors have found that agents that boost expression of Bcl2-associated agonist of cell death (BAD) can enhance the cytotoxicity of chemotherapeutic drugs in many cancer cell lines. The increase in overall cytotoxicity is independent of the cytotoxicity of agents that increase expression of BAD, which by themselves have no or minimal cytotoxic effects.

According to a first aspect of the present invention, there is provided an agent that increases expression of BAD for use in the treatment of cancer in combination with a chemotherapeutic agent.

According to a second aspect of the present invention, there is provided a method of selecting a subject having cancer for treatment with an agent that increases expression of BAD, the method comprising the steps of (a) obtaining a sample from the subject suspected of needing same, (b) measuring a concentration of BAD in the sample, and (c) comparing the measured concentration of BAD with a reference value, wherein if the subject has a concentration of BAD that is approximately equal to or less than the reference value, then the subject is selected for administration of an agent that increases expression of BAD.

According to a third aspect of the present invention, there is provided a method of screening for an agent that increases expression of BAD for use according to the first aspect of the present invention, the method comprising the steps of (a) culturing a cell with a test agent, (b) measuring the concentration of BAD after culturing with the test agent, and (c) comparing the fold change in expression of BAD between the cell and a control value, wherein if the fold change in expression of BAD is at least 10%, the agent is identified as an agent for use according to the first aspect of the present invention.

According to a fourth aspect of the present invention, there is provided a method for treating a subject having cancer, comprising the step of administering an anticancer agent, wherein the treated subject exhibits a 10% increase in the expression level of BAD in cancer cells compared to a control.

The invention is further defined by reference to the following figures:

FIG. 1a shows the effect of different concentrations of 6-β-naltrexol on the expression levels of BAD, p21, pAKT, cyclin B1 and CDK1 in HCT116 cells. Ibid. Figure 2 shows the cytostasis (a and b) and cytotoxicity (c and d) of A549 cells (a and c) and HCT116 cells (b and d) upon co-administration of 10 nM or 10 μM 6-β-naltrexol in combination with GEM or OXP. Control samples of cells were treated without 6-β-naltrexol. Ibid. FIG. 3 shows a dose-dependent increase in expression levels of BAD in breast cancer cells in response to different concentrations of 6-β-naltrexol. Figure 4 shows cytostasis (a) and cytotoxicity (b) of breast cancer cell lines upon treatment with 10 nM (6BN(n)) or 10 μM 6-β-naltrexol (6BN(u)), alone or in combination with gemcitabine (GEM) or oxaliplatin (OXP). Results from control cells treated with no drug (UN), GEM or OXP alone are also shown.

The present invention is based on the discovery that agents that increase the expression of BAD increase the sensitivity of cancer cells to chemotherapeutic agents. This is exemplified by the use of 6-β-naltrexol. Thus, co-administration of 6-β-naltrexol with chemotherapeutic agents can boost the therapeutic efficacy of anti-cancer treatment regimens. By increasing therapeutic efficacy, 6-β-naltrexol can obviate the need to implement particularly aggressive therapeutic strategies, which often exhibit adverse side effects. Furthermore, the effect of increased efficacy can rescue certain chemotherapeutic agents that have shown limited efficacy in treating certain cancers.

The inventors have found that the activity of 6-β-naltrexol is independent of any cytotoxic activity. Thus, while 6-β-naltrexol alone has negligible therapeutic effect, the drug can be used to enhance the cytotoxicity of co-administered chemotherapeutic agents. The ability of 6-β-naltrexol to enhance therapeutic activity was observed in at least three independent cell lines using at least two separate classes of chemotherapeutic agents, thus suggesting that the effects of 6-β-naltrexol can be utilized to boost the therapeutic efficacy of multiple chemotherapeutic agents for use in the treatment of multiple cancers.

Without wishing to be bound by theory, 6-β-naltrexol appears to alter the phenotype of cancer cells in a way that increases the sensitivity of the cells to chemotherapeutic drugs. One particular marker whose expression changes in response to 6-β-naltrexol is BAD. Thus, the expression level of BAD, or as used interchangeably herein, "level," can be used to determine that cancer cells will be sensitive to subsequent administration of anti-cancer drugs. Thus, it is envisioned that any agent that increases BAD expression can be used to increase the sensitivity of cancer cells to chemotherapeutic drugs.

The invention can be further understood with reference to the following definitions.
As used herein, "6-β-naltrexol" refers to 17-(cyclopropylmethyl)-4,5-epoxymorphinan-3,6 beta,14-triol (CAS No. 49625-89-0). "6-β-naltrexol" may be included in the pharmaceutical formulation as a pharma- ceutically acceptable salt, solvate, hydrate, stereoisomer, or clathrate thereof. 6-β-naltrexol is the major active metabolite of naltrexone.

As used herein, "increased expression" and synonyms refer to an increase in the expression level (i.e., "level") of a particular cellular biomarker upon administration of 6-β-naltrexol. An increase in the expression level of a particular cellular biomarker indicates that the cancer cells received the desired response upon administration of the first agent and are therefore more sensitive to the cytotoxic effects of the chemotherapeutic agent. The expression level of a biomarker can be measured in a sample using any number of analytical methods available to one of skill in the art, including, but not limited to, gel electrophoresis, Western blot analysis, 2D-PAGE, column chromatography, ribosome profiling, or mass spectrometry. An increased level of expression can be determined by comparing the expression level of the biomarker before and after administration of 6-β-naltrexol. The expression level of the biomarker before administration of 6-β-naltrexol can be referenced as a control. In some examples, for measurement of a particular biomarker, it may be desirable to purify the biomarker from the cellular environment before analyzing the expression level. The type of purification method used will depend on the type of biomarker being analyzed. In general, biomarker purification techniques are well known to those of skill in the art, and a non-exhaustive list of techniques can be found in Protein Purification Techniques, Second Edition, Simon Roe, Oxford University Press (2001), which is incorporated herein in its entirety.

A biomarker of particular interest in the context of the present invention is BAD. BAD is a Bcl2-associated agonist of cell death.
BAD, also known as BAD1, is a member of a family of BH3-only proapoptotic proteins that initiate cell death upon activation, and whose activity is largely controlled by post-translational modifications that integrate various cell survival and death signals (Danial 2009). BAD specifically promotes apoptosis through the binding and neutralization of its antiapoptotic partners BCL-2, BCL- _XL , and BCL-W, which are located in mitochondria, where BAD is able to withdraw growth factor survival signals (from the cytosol).
The present invention shows that levels of BAD are increased in response to administration of 6-β-naltrexol. Any molecule that increases the expression level of BAD is contemplated to be encompassed within this aspect of the invention. Preferably, any agent in any dosing regimen that increases the expression level of BAD by at least 5%, or at least 10%, or at least 20%, or at least 30%, or at least 40% compared to a control is encompassed within the embodiments of the invention. More preferably, the agent increases the expression level of BAD by at least 10% compared to a control. More preferably, the agent increases the expression level of BAD by 25-100% compared to a control.

As used herein, the term "subject" refers to any animal (e.g., a mammal), including but not limited to humans, non-human primates, dogs, cats, rodents, etc., that is the recipient of a treatment in which an agent that increases expression of BAD is used in accordance with the present invention. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

As used herein, "chemotherapeutic agent" has its conventional meaning as used in the art. The terms "chemotherapeutic agent" and "anticancer agent" are used synonymously in this invention.

According to a first aspect of the present invention, there is provided an agent that increases expression of BAD for use in the treatment of cancer in combination with a chemotherapeutic agent, where "in combination" means that the agent forms part of an anti-cancer treatment regimen together with the chemotherapeutic agent.

In certain embodiments, the agent is administered in an amount sufficient to increase the expression of BAD by at least 5%, at least 10%, at least 20%, or at least 30%, or at least 40% compared to a control. Preferably, the agent is administered in an amount sufficient to increase the expression level of BAD by at least 10% compared to a control. In certain embodiments, the control is the expression level of BAD in a sample obtained from the subject prior to administration of the agent. One of skill in the art would be able to determine such an effective amount by performing routine laboratory experiments to measure the increase in the expression level of BAD in response to administration of increasing amounts of the agent. In certain embodiments, the biological sample obtained from the subject for use in the method is blood, plasma, serum, lymph, tissue, or cells derived from a tissue sample. Preferably, the sample is obtained by tumor biopsy of the subject. Conventional techniques for obtaining any of the above biological samples from a subject are well known to those of skill in the art.

Preferably, the agent that increases the expression of BAD is selected from the list consisting of 6-β-naltrexol, naloxone, methylnaltrexone or a pharma- ceutically acceptable salt thereof. Preferably, the agent is 6-β-naltrexol or a pharma-ceutically acceptable analog thereof.

In certain embodiments, when the agent is 6-β-naltrexol, the 6-β-naltrexol is administered in an amount effective to increase the plasma concentration of 6-β-naltrexol by at least 0.34 ng/ml, at least 3.4 ng/ml, at least 34 ng/ml, or at least 340 ng/ml. In certain embodiments, the 6-β-naltrexol is administered in an amount effective to increase the plasma concentration of 6-β-naltrexol to within the range of 0.3 to 3,400 ng/ml, preferably 34 to 3,400 ng/ml, and more preferably 340 to 3,400 ng/ml. Amounts effective to achieve such amounts can be determined using any number of conventional techniques known to those of skill in the art. For example, one of skill in the art can perform mass spectrometry on a plasma sample obtained from a subject to measure the increase in the concentration of 6-β-naltrexol in the sample following administration of an amount of 6-β-naltrexol. An effective amount is that amount determined to produce the desired increase in plasma concentration.

As used herein, the terms "treat" and "treatment" and "treating" refer to both 1) therapeutic treatments that cure, slow, and/or halt the progression of a diagnosed pathological condition or disorder, and 2) prophylactic or preventative treatments that prevent and/or slow the onset of the targeted pathological condition or disorder. Thus, those in need of treatment include those already suffering from the disorder, those prone to suffering from the disorder, and those in whom the disorder is to be prevented. In some instances, a tumor/cancer may be said to be successfully "treated" according to the present invention if the subject exhibits one or more of the following: a reduction or complete absence of cancer cells in number, a reduction in size of the tumor, inhibition or absence of cancer cell invasion into surrounding tissues, including spread of cancer to soft tissues and bone, inhibition or absence of tumor metastasis, inhibition or absence of tumor growth, reduced morbidity or mortality, a reduction in tumorigenicity, tumorigenic frequency, or tumorigenic potential of the tumor, a reduction in the number or frequency of cancer stem cells in the tumor, differentiation of tumorigenic cells to a non-tumorigenic state, or a combination of several effects.

As used herein, the term "tumor/cancer" refers to any neoplasm of tissue resulting from excessive cell growth, proliferation and/or survival, either benign (non-cancerous) or malignant (cancerous), including pre-cancerous, lesions. The terms "tumor/cancer" and "neoplasm" may be used interchangeably. The term "tumor cell" refers to a cell or cells derived from a tumor/cancer.

As used herein, the term "cancer cell" refers to a cell or immortalized cell line derived from a tumor or cancer.

In certain embodiments, the agent that increases expression of BAD may be administered simultaneously, separately or sequentially with the chemotherapy agent.

As used herein, the terms "co-administration" or "concurrently" or "concurrently," "sequentially," or "separately" mean that the administration of the agent that increases expression of BAD and the chemotherapeutic agent occurs as part of the same treatment regimen.

"Concurrent" administration, as defined herein, includes administration of the agent that increases expression of BAD and the chemotherapeutic agent within about 2 hours or about 1 hour or less of each other, and more preferably simultaneously.

"Separate" administration, as defined herein, includes administration of the agent that increases expression of BAD and the chemotherapeutic agent about 12 hours, or about 8 hours, or about 6 hours, or about 4 hours, or about 2 hours or more apart.

"Sequential" administration, as defined herein, includes administering the agent that increases expression of BAD and the chemotherapeutic agent in multiple divided doses and/or doses and/or on separate occasions, respectively. The agent that increases expression of BAD may be administered to the subject before or after administration of the chemotherapeutic agent. Alternatively, the chemotherapeutic agent may continue to be administered to the subject after treatment with the agent that increases expression of BAD has ceased.

In certain embodiments, the chemotherapy agent is administered after the agent that increases expression of BAD is administered.

In particular embodiments, the chemotherapeutic agent is administered when the expression level of BAD increases by at least 5%, or at least 10%, or at least 20%, or at least 30%, or at least 40% relative to a control. Preferably, the chemotherapeutic agent is administered when the expression level of BAD increases by at least 10% relative to a control.

In certain embodiments, the drug and the chemotherapy agent are administered simultaneously.

Further in accordance with the first aspect, the chemotherapeutic agent may be selected from the group consisting of PI3-kinase inhibitors, AKT inhibitors, taxanes, antimetabolites, alkylating agents, cell cycle inhibitors, topoisomerase inhibitors and cytotoxic antibodies. The chemotherapeutic agent may be administered in any conventional manner, the method of administration being highly dependent on the anticancer agent used. Thus, administration by inter alia parenteral, oral, sublingual, nasal and/or pulmonary routes is envisaged.

When the chemotherapeutic agent is a PI3-kinase inhibitor, suitable examples include wortmannin, LY294002, demethoxyviridin, IC87114, NVP-BEZ235, BAY 80-6946, BKM120, GDC-0941, GDC-9080, combinations thereof, and pharma- ceutically acceptable salts, solvates, hydrates, stereoisomers, clathrates, and prodrugs of any of the above, but are not limited to these.

When the chemotherapeutic agent is an AKT inhibitor, suitable examples include, but are not limited to, MK-2206, GSK690693, perifosine, PHT-427, AT7867, honokiol, PF-04691502, combinations thereof, and pharma- ceutically acceptable salts, solvates, hydrates, stereoisomers, clathrates, and prodrugs of any of the above.

When the chemotherapeutic agent is a taxane, suitable examples include, but are not limited to, paclitaxel and docetaxel, combinations thereof, and pharma- ceutically acceptable salts, solvates, hydrates, stereoisomers, clathrates, and prodrugs of any of the above.

When the chemotherapeutic agent is an antimetabolite, suitable examples include, but are not limited to, methotrexate, 5-fluorouracil, capecitabine, cytosine arabinoside (cytarabine), gemcitabine, 6-thioguanine, pentostatin, azathioprine, 6-mercaptopurine, fludarabine and cladribine, including combinations thereof, and pharma- ceutically acceptable salts, solvates, hydrates, stereoisomers, clathrates and prodrugs of any of the above. Gemcitabine is a particularly preferred antimetabolite. By way of example, gemcitabine may be administered at a dose (per administration) of 800-1200 mg/ ^m2 , preferably 900-1100 mg/ ^m2 , e.g., about 1000 mg/ ^m2 or 1000 mg/ ^m2 .

When the chemotherapeutic agent is an alkylating agent, suitable examples include, but are not limited to, mechlorethamine, cyclophosphamide, ifosfamide, trofosfamide, melphalan (L-sarcolysin), chlorambucil, hexamethylmelamine, thiotepa, busulfan, carmustine (BCNU), streptozocin (streptozotocin), dacarbazine (DTIC; dimethyltriazenoimidazole carboxamide), temozolomide, and oxaliplatin, including combinations thereof, and pharma- ceutically acceptable salts, solvates, hydrates, stereoisomers, clathrates, and prodrugs of any of the above. Cyclophosphamide and oxaliplatin are particularly preferred alkylating agents. By way of example, oxaliplatin may be administered at a dose (per administration) of 65-105 mg/ ^m2 , preferably 75-95 mg/ ^m2 , such as about 85 mg/ ^m2 or 85 mg/ ^m2 . By way of example, cyclophosphamide may be administered at a dose (per administration) of up to 1800 mg/ ^m2 , such as 400-1800 mg/ ^m2 .

When the chemotherapeutic agent is a cell cycle inhibitor, suitable examples include epothilones, vincristine, vinblastine, staurosporine/UCN-01, 17AAG, XL844, CHIR
-124, PF-00477736, CEP-3891, flavopiridol, berberine, P276-00, terameprocol, isoflavone daidzein, BI2536, BI6727, GSK461364, cyclaporin, ON-01910, NMS-P937, TAK-960, ispinesib, monastrol, AZD4877, LY2523355, ARRY-520, MK-0731, SB743921, GSK923295, lonafarnib, proTAME, bortezomib, MLN9708, ONX0912, CEP-18770, combinations thereof, and pharma- ceutically acceptable salts, solvates, hydrates, stereoisomers, clathrates, and prodrugs of any of the above, but are not limited to these. Particularly suitable examples of cell cycle inhibitors include hesperadin, ZM447439, VX-680, MLN-8054, PHA-739358, AT-9283, AZD1152, MLN8237, ENMD2076, SU6668, including combinations thereof, as well as other inhibitors of Aurora kinase, and including, but not limited to, pharma- ceutically acceptable salts, solvates, hydrates, stereoisomers, clathrates and prodrugs of any of the above.

In certain embodiments, the chemotherapy agent is an antimetabolite, preferably gemcitabine.

In certain embodiments, the chemotherapy agent is an alkylating agent, preferably oxaliplatin.

The present invention may be used to treat cancers including sarcomas, carcinomas, adenocarcinomas, melanomas, myelomas, blastomas, gliomas, lymphomas, or leukemias. Representative cancers include, for example, carcinomas, sarcomas, adenocarcinomas, melanomas, neurogenic (blastomas, gliomas), mesotheliomas, and reticuloendothelial, lymphoid, or hematopoietic neoplastic disorders (e.g., myelomas, lymphomas, or leukemias). In certain embodiments, the tumor or cancer includes lung adenocarcinoma, lung cancer, diffuse or interstitial gastric cancer, colon adenocarcinoma, prostate adenocarcinoma, esophageal cancer, breast cancer, pancreatic adenocarcinoma, ovarian adenocarcinoma, adenocarcinoma of the adrenal gland, endometrial adenocarcinoma, or uterine adenocarcinoma.

Tumors and cancers include benign, malignant, metastatic and non-metastatic types, including progressive, worsening, stabilized or remitted tumors, cancers or metastases of any stage (I, II, III, IV or V) or grade (G1, G2, G3, etc.). Cancers that may be treated according to the present invention include, but are not limited to, bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestines, gums, head, kidney, liver, lung, nasopharynx, neck, ovaries, prostate, skin, stomach, testes, tongue or uterus. Preferably, the cancer is selected from prostate cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colon cancer, pancreatic cancer, brain cancer, hepatocellular carcinoma, lymphoma, leukemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, head and neck cancer, skin cancer and soft tissue sarcoma and/or other forms of cancer. The tumor may be a metastatic or malignant tumor.

In certain embodiments, the cancer being treated is selected from the list consisting of lung cancer, colon cancer, breast cancer, pancreatic cancer, lymphoma or glioma.

In certain embodiments, the cancer being treated is preferably breast cancer.

In certain embodiments, the cancer being treated is lung cancer or colon cancer. Preferably, the cancer being treated is colon cancer. Preferably, the cancer being treated is colon cancer.

In a second aspect of the present invention, there is provided a method for selecting a subject having cancer for treatment with an agent that increases the expression level of BAD, the method comprising the steps of: (a) obtaining a sample from the cancer subject suspected of needing same; (b) measuring the concentration of BAD in the sample; and (c) comparing the measured concentration of BAD with a reference value, wherein if the subject's concentration is approximately equal to or less than the reference value, the subject is selected for administration of the agent that increases the expression of BAD.

The term "approximately" is used herein to provide strict support for the exact value that the term precedes, and for a value that is near or around the value that the term precedes. In determining whether a value is near or around a specifically recited value, the near or around unrecited value may be a value that, in the context in which it occurs, provides a substantial equivalent to the specifically recited value. For example, "approximately" may mean that the value is within 1%, 2%, or 5% of the reference value.

In a particular embodiment of the second aspect, the method can be used to monitor the therapeutic effectiveness of an agent that increases expression of BAD, and comprises carrying out steps (a) to (c) according to the second aspect of the invention after the subject has been administered the agent.

By monitoring the expression level of BAD after administration of the agent, the subject can then be administered the chemotherapy agent once it has become sensitized to the chemotherapy agent. If the BAD concentration after administration of the agent is at least 5%, at least 10%, at least 20%, at least 30% or at least 40%, preferably at least 10%, higher than the baseline, the subject can be selected for administration of the chemotherapy agent. Alternatively, if the BAD concentration is 5%, 10%, 20%, 30% or 40% lower than the baseline, the subject is selected for re-administration of the agent that increases BAD expression. Preferably, when the BAD concentration after administration of the agent is 10% lower than the baseline, the subject is selected for re-administration of the agent that increases BAD expression.

In accordance with the second aspect of the invention, the phrases "reference value" and "control value" are used interchangeably herein. A "reference" value for use in the present method can be the expression level of BAD measured from a biological sample obtained from a healthy subject. As used herein, a "healthy subject" refers to a subject not suffering from cancer. The reference value can be determined by measuring the expression level of BAD in a sample obtained from a healthy individual at the time of carrying out the method of the second aspect of the invention. Alternatively, the reference value can be a defined value by previously measuring the expression level of BAD in an equivalent sample obtained from a healthy individual. When monitoring the therapeutic efficacy of an agent that increases the expression of BAD, the reference value can be derived from a healthy individual, or the reference value can be a measured BAD concentration in a sample previously obtained from the subject, i.e., the reference value can be the expression level of BAD in a sample obtained from the subject before administration of the agent.

In certain embodiments, the biological sample obtained from a subject for use in the method is blood, plasma, serum, lymph, tissue, or cells derived from a tissue sample. Preferably, the sample is obtained from a tumor biopsy of the subject. Conventional techniques for obtaining any of the above biological samples from a subject are well known to those of skill in the art.

In certain embodiments, the expression level of BAD is measured by any method selected from the list consisting of Western blot, mRNA expression analysis, ribosome profiling, flow cytometry, and mass spectrometry. The expression level of BAD may also be determined using other conventional analytical methods known to those skilled in the art.

In certain embodiments, the agent that increases the expression of BAD is selected from the group consisting of 6-β-naltrexol, naloxone, methylnaltrexone, or a pharma- ceutically acceptable salt thereof. Preferably, the agent that increases the expression of BAD is 6-β-naltrexol, or a pharma- ceutically acceptable salt thereof.

In certain embodiments, the chemotherapeutic agent is selected from the group consisting of PI3-kinase inhibitors, AKT inhibitors, taxanes, antimetabolites, alkylating agents, cell cycle inhibitors, topoisomerase inhibitors and cytotoxic antibodies.

In certain embodiments, the cancer subject has a cancer selected from the list consisting of lung cancer, colon cancer, breast cancer, pancreatic cancer, lymphoma, or glioma. Preferably, the cancer subject has lung cancer or colon cancer. In certain embodiments, the cancer is colon cancer. In certain embodiments, the cancer is breast cancer.

According to a third aspect of the present invention, there is provided a method of screening for an agent that increases the expression of BAD for use according to any embodiment of the first aspect of the present invention, the method comprising the steps of (a) culturing a cell with a test agent, (b) measuring the concentration of BAD after culturing with the test agent, and (c) comparing the increase in the expression level of BAD between the cell and a control value, wherein if the increase in the expression level of BAD is at least 5%, or at least 10%, or at least 20%, or at least 30%, or at least 40% compared to the control, the agent is identified as an agent for use according to the first aspect of the present invention. Preferably, there is provided a method wherein if the agent increases the expression level of BAD by at least 10% compared to the control, the agent is identified as an agent for use according to any embodiment of the first aspect of the present invention.

To carry out the method of the third aspect of the invention, one skilled in the art may utilize any number of standard cell culture techniques routinely used in in vitro drug screening protocols. For example, a multi-well in vitro cell culture format may be used, allowing multiple candidate drugs to be screened simultaneously at multiple concentrations. From such a format, one skilled in the art may then determine the most appropriate drug by analyzing how expression of BAD increases as a function of drug concentration. To determine the expression level of BAD, any number of methods known in the art may be performed by one skilled in the art, including but not limited to RT-PCR, Western blotting, immunohistochemistry and suitable derivatives of the above. The fold change in expression may be determined with reference to a control value derived from a population of cells cultured with or without any agent, where the agent is a molecule known not to increase the expression level of BAD. Suitable agents will be known to one skilled in the art, or a suitable agent may be an agent that does not increase expression of BAD as determined from the screening method. Alternatively, the control value may be a defined value corresponding to the endogenous level of BAD expression in the population of cells used in the assay.

In an embodiment of the third aspect of the invention, the cells are or are derived from an immortalized cell line, preferably of human origin. An "immortalized" cell line refers to a cell population that proliferates indefinitely due to mutations and avoids normal cellular senescence. For example, cells include SH-SY5Y, Hep-G2, HEK 293, RAW 264.7, HeLa, MRC-5, A2780, CACO-2, THP 1, A549, PD 30, MCF7, SNL 76/7, C2C12, Jurkat E6.1, U937, L929, 3T3 L1, HL60, PC-12, HT29, OE33, OE19, NIH 3T3, MDA-MB-231, K562, U-87 MG, PD-25, A2780cis, B9, CHO-K1, MDCK, 1321N1, A431, ATDC5, HUVEC, Vero, Fao, J774A. 1, MC3T3-E1, J774.2, PNT1A, U-2 OS, HCT 116, MA104, BEAS-2B, NB2-11, BHK 21, NS0, Neuro 2a, T47D, 1301, PNT2, PC-3, TF1, COS-7, MDCK, NCI-322, SK, N. SH, LNCaP. FGC, OE21, PSN1, ISHIKAWA, MF
The cell may be or may be derived from E-280, MG-63, RK 13, EoL-1 cell, VCaP, tsA201, CHO, HT 1080, PANC-1, Saos-2, SK-OV-3, COV434, Hep 3B, A375, AGS, CAKI 2, COLO 205, COR-L23, IMR 32, QT 35, WI 38, HMVII, HT55 or TK6 cells.

According to a fourth aspect of the present invention, there is provided a method for treating a subject having cancer, comprising the step of administering an anticancer agent, wherein the treated subject exhibits a 10% increase in the expression level of BAD in cancer cells compared to a control.

This aspect of the invention is based on the discovery that tumor cell lines with increased expression levels of BAD are more sensitive to the effects of anti-cancer drugs. As used herein, "sensitized" refers to an increased sensitivity of cancer cells to cytotoxicity in response to administration of an anti-cancer drug, whereby the increased "sensitivity" is due to an increased expression level of BAD compared to a control. An increased expression level of BAD can be an inherent characteristic of tumor cells, or the increased expression level can be induced by administering an agent that increases expression of BAD. The increased expression level can be determined relative to a basal level of BAD expression in non-tumor cells of a subject receiving the anti-cancer drug. In this example, the basal level of expression in the non-tumor cells is referred to as the control. Alternatively, the basal level of BAD expression can be a default value derived from the expression level of BAD in non-tumor cells of a healthy subject.

In a fourth aspect, there is provided a method of treating a subject having cancer, comprising administering an anti-cancer agent to the subject, wherein the treated subject exhibits a 10% increase in the expression level of BAD in cancer cells compared to a control. The expression level of BAD in the subject may be increased by administering an agent according to the first aspect of the invention.

In a fifth aspect of the present invention, there is provided the use of an agent that increases expression of BAD in the manufacture of a medicament for treating cancer, the medicament being administered in combination with a chemotherapeutic agent.

In further embodiments of both the fourth and fifth aspects of the invention, the method or use thereof has the same optional and preferred features as applicable to the first aspect of the invention.

For use in the present invention, a pharmaceutical composition is provided that includes 6-β-naltrexol or an analogue thereof, or a pharma- ceutically acceptable salt of either. The pharmaceutical composition may be provided as an oral aqueous solution, a caplet, a capsule, an injectable, an insoluble formulation, a suppository, a lozenge, or a tablet. In a particular embodiment, the pharmaceutical composition is provided as an oral dosage form, specifically a tablet.

As used herein, the term "pharmaceutical composition" refers to a mixture that includes, for example, a particular amount, e.g., a therapeutically effective amount, of a therapeutic compound or compounds in a pharma- ceutical acceptable carrier for administration to a mammal, such as a human, to treat a disease.

As used herein, the term "pharmacologically acceptable" refers to compounds, materials, compositions and/or dosage forms that are, within the bounds of medical good sense, suitable for contact with mammalian, and particularly human, tissues and that exhibit a reasonable benefit/risk ratio without excessive toxicity, irritation, allergic response and other significant complications.

The term formulation is intended to include a mixture of active ingredient(s) with an encapsulating material as a carrier to provide a solid dosage form, and the carrier is therefore related to the formulation since the active compound is surrounded by the carrier (with or without other carriers). Similarly, cachets are included. Tablets, powders, cachets and capsules can be used as solid dosage forms suitable for oral administration.

The pharmaceutical preparation can be in unit dosage form. In such dosage form, the composition is divided into a number of unit doses containing appropriate amounts of the active ingredient or ingredients. The unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparation, for example packeted tablets, capsules, and powders in a vial or ampoule. The unit dosage form can also be a capsule, cachet, or tablet itself, or it can be the appropriate number of any of these packaged forms.

In one embodiment, the 6-β-naltrexol product is utilized in a solid oral dosage form comprising a therapeutically effective amount of 6-β-naltrexol in the compositions of the present invention, for example, about 0.01-50 mg, preferably about 0.01-40 mg, and most preferably about 0.01-20 mg of 6-β-naltrexol product per tablet, and may be about 0.01 mg, about 0.05 mg, about 0.1 mg, about 0.3 mg, about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 5 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, or about 50 mg of 6-β-naltrexol product per tablet. In certain embodiments, the composition includes an appropriate dosage of the 6-β-naltrexol product to account for the degradation, if any, of the 6-β-naltrexol product. In certain embodiments, the composition includes 3-4.5 mg of the 6-β-naltrexol product.

The pharmaceutical composition may be provided as a mixture of a 6-β-naltrexol product and a combination of pharma- ceutically acceptable excipients. As used herein, the term "excipient" refers to a pharma- ceutically acceptable ingredient commonly used in the pharmaceutical arts for preparing solid oral dosage formulations. Examples of excipient classes include, but are not limited to, binders, disintegrants, lubricants, glidants, stabilizers, fillers, and diluents. The amount of each excipient used may vary within ranges conventional in the art. The following references, all of which are incorporated herein by reference, disclose techniques and excipients used to formulate oral dosage forms: The Handbook of Pharmaceutical Excipients, 4th edition, Rowe et al., Eds. , American Pharmaceuticals Association (2003); and Remington: the Science and Practice
of Pharmacy, 20th edition, Gennaro, Ed., Lippincott Williams & Wilkins (2000).

Suitable excipients include magnesium carbonate, magnesium stearate, talc, lactose, lactose monohydrate, sugar, pectin, dextrin, starch, tragacanth, microcrystalline cellulose, methylcellulose, sodium carboxymethylcellulose, corn starch, colloidal anhydrous silica, titanium dioxide, low melting waxes, cocoa butter, and the like.

In other embodiments, the pharmaceutical composition includes at least one excipient.

The invention is illustrated by the following non-limiting examples.

Example 1
Measurement of BAD expression levels after administration of 6-β-naltrexol To measure the effect of 6-β-naltrexol or naltrexone on the expression levels of BAD, pAKT, p21, cyclin B1 and CDK1, HCT116 colon cancer cells were seeded in 6-well plates at a concentration of 2× ¹⁰⁵ cells/well and allowed to attach overnight. Cells were then cultured for an additional 48 hours in the presence of naltrexone or 6-β-naltrexol at concentrations of 10 nM or 10 μM. Cells were then harvested and processed using standard immunoblotting techniques to measure BAD, or pAKT, p21, cyclin B1 and CDK1.

Results showed that 10 μM 6-β-naltrexol increased BAD expression levels by more than 10% in HCT116 cells, but both concentrations of naltrexone and 10 nM 6-β-naltrexol had negligible effects on BAD expression levels.

Example 2
Increased BAD expression levels boost the efficacy of anticancer drugs The impact of combining 6-β-naltrexol with other chemotherapeutic drugs was tested by culturing cells according to a treatment schedule that included two treatment phases. The first phase involved stimulation with 10 nM 6-β-naltrexol or 10 μM 6-β-naltrexol for 48 h before treatment with the other drug for an additional 48 h. A549 and HCT116 were seeded at a concentration of 2 × ¹⁰⁵ cells/well in 6-well plates and allowed to attach overnight. After 48 h, the medium was removed and the cells were gently rinsed with drug-free medium. Fresh culture medium containing gemcitabine (GEM) or oxaliplatin (OXP) was then added to the cells. The chemotherapy drug concentrations used were approximately ¼ IC50, as previously established (Liu WM, Fowler DW, Smith P, Dalgleish AG. Pre-treatment with chemotherapy can enhance the antibody and immunogenicity of tumors by promoting adaptive immune responses. Br J Cancer. 2010 Jan 5;102(1):115-23. doi: 10.1038/sj.bjc.6605465.). The cells were then left for an additional 48 hours after which cell number or viability was assessed by cell counting using trypan blue dye as a method to distinguish between live and dead cells. There was a decrease in cell number, but no associated decrease in cell viability, indicating cell division arrest.

Experiments show that 6-β-naltrexol increases the cytotoxic effects of both chemotherapeutic agents when added in an amount effective to increase the expression level of BAD by at least 10%. This effect was observed without an independent increase in the cytotoxicity caused by administering 6-β-naltrexol alone (Figures 2c and d, 10 nM 6BN-0 or 10 μM 6BN-0). Furthermore, when the expression level of BAD is increased to a greater extent (i.e., by administering a large dose of 6-β-naltrexol), the cytotoxic effect is greatly enhanced.

Thus, experiments suggest that agents that increase the expression of BAD can enhance the therapeutic efficacy of certain anticancer drugs.

Example 3
Determining the effect of 6BN on cancer cell lines Similar experiments to those performed on A549 and HCT116 cells were also completed in the breast cancer cell line MCF-7 to assess the effect of 6BN on protein expression by Western blot, with results showing that 6BN increased the expression of the pro-apoptotic protein BAD in a dose-dependent manner (Figure 3). The effect of combining 6BN with the chemotherapy drugs gemcitabine or oxaliplatin was measured by measuring the effect of 6BN (10 nM or 10 μM) on MCF-7 cells.
6BN at 10 uM was tested by co-incubation with gemcitabine (0.5 uM) or oxaliplatin (0.5 uM). Cell numbers and viability were then assessed after 48 hours using trypan blue exclusion as a means of distinguishing live/dead cells. Results showed that 10 uM 6BN in combination with any chemotherapy produced the greatest effect on cell viability (Figure 4).

Claims (14)

A pharmaceutical composition in solid dosage form comprising 0.01 mg to 50 mg per tablet of an agent that increases expression of BAD (Bcl2 associated cell death agonist), in combination with a chemotherapeutic agent, for treating cancer, wherein the agent is 6-β-naltrexol or a pharma- ceutical agent is an antimetabolite or an alkylating agent, and the cancer is selected from the list consisting of lung cancer, colon cancer, and breast cancer . The pharmaceutical composition of claim 1, wherein the agent is administered in an amount sufficient to increase expression of BAD by at least 5% relative to a control. The pharmaceutical composition of claim 2, wherein the agent is administered in an amount sufficient to increase expression of BAD by at least 40% relative to a control. The pharmaceutical composition according to any one of claims 1 to 3, wherein the 6-β-naltrexol is administered in an amount effective to increase the plasma concentration of 6-β-naltrexol to 0.34 to 3,400 ng/ml. The pharmaceutical composition according to claim 4, wherein the 6-β-naltrexol is administered in an amount effective to increase the plasma concentration of 6-β-naltrexol to 34-3,400 ng/ml. The pharmaceutical composition according to claim 5, wherein the 6-β-naltrexol is administered in an amount effective to increase the plasma concentration of 6-β-naltrexol to 340-3,400 ng/ml. The pharmaceutical composition according to any one of claims 1 to 6, which is administered separately, sequentially or simultaneously with a chemotherapy drug. The pharmaceutical composition of claim 7, wherein the drug and the chemotherapeutic agent are administered sequentially or separately. The pharmaceutical composition according to claim 7 or 8, wherein the chemotherapeutic agent is administered after the pharmaceutical composition containing an agent that increases expression of BAD is administered. The pharmaceutical composition according to any one of claims 7 to 9, wherein the chemotherapeutic agent is administered when the expression level of BAD increases by at least 5% relative to a control. The pharmaceutical composition of claim 7, wherein the drug and the chemotherapeutic agent are administered simultaneously. The pharmaceutical composition according to any one of claims 7 to 11 , wherein the chemotherapeutic agent is gemcitabine. The pharmaceutical composition according to any one of claims 1 to 12 , wherein the cancer is lung cancer or colon cancer. The pharmaceutical composition according to any one of claims 1 to 12 , wherein the cancer is breast cancer.

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